Comparing Method for Expression Amount of the Same Gene from Different Sources by Base Sequence Measurement

ABSTRACT

The present invention relates to an analysis method used to quantitatively compare the expression levels of the same gene from different sources. The method of the present invention can be used to quantitatively compare the gene expression level difference of the same gene of tissues or cells from different sources, making use of the quantitative characteristics of bioluminescent assay and the principle of adding different deoxyribonucleic acids (dNTP) one by one. The concrete steps are: reverse transcript the messenger ribonucleic acids (mRNA) from different sources into cDNA, and label a segment of source specific sequence in cDNA from each source; mix the labeled cDNA of different sources into one tube and use it as the substrate of polymerase chain reaction (PCR); PCR amplification is performed using the same common primer and a gene-specific primer; Detect the base sequence by bioluminescent assay, wherein the base type represents the different gene source, and the signal intensity of each base represents the gene expression level from each source. This method has a significant meaning for the screening of disease-related genes, clinical early diagnosis and the preparation of specific medicine for the treatment of disease.

CROSS REFERENCE TO THE RELATED PATENT APPLICATION

This application claims the priority of the Chinese application No.200410062751.2, filed on Jul. 9, 2004.

FIELD OF THE INVENTION

The present invention relates to a method used to quantitatively comparethe relative expression level of the same gene of tissues or cells fromdifferent sources. Specifically, it is a base sequencing method for thedetermination of the relative content of each DNA fragment in themixture of DNA fragments labeled by different base sequences and all thedeterminations can be carried out at the same time.

BACKGROUND OF THE INVENTION

With the progress in molecular biology and analytical apparatus, thesequencing work of human genome project (HGP) has already been finished.As the structure of the whole human genome is clarified, the next stepis to analyze gene functions coded in genomes^([1]), gene functionanalysis includes the understanding the distribution of the genetranscription products mRNA and the quantity, distribution and functionof proteins (the translated products of mRNA) in a cell or differentorgans in a body. We can look for and find the disease-related genes bycomparing the gene expression levels between healthy persons andpatients, and further make them used in clinical early diagnosis^([2]).In drug screening process, the target of drug can be found by detectingthe relative gene levels of the administration group and the untreatedgroup, and further look for and prepare the specific medicine for thetreatment of disease^([3]). Therefore, the differential analysis of geneexpression level has become one of the main tasks of the“post-sequencing age”. The developed countries have invested a lot ofmaterial resources and money to rank top in this field and furthermonopoly the technology. At present the major analysis methods for thegene expression level comparison are: SAGE method^([4]), RT-PCR (reversetranscription-polymerase chain reaction) method^([5]) and microarray(gene chip)^([6]), etc. But these methods still have some drawbacks:only the gene expression levels of two individuals can be compared at atime; the prices of apparatus are very high; the operation is complexand the quantitative characteristics are bad, etc. For example, in thecase of SAGE method, detection is very tedious and there are too manysteps so it is hard to control, in addition, the cost is also very high,all these lead to its small popularity. RT-PCR method needs specialapparatus and internal standard, its detection is also tedious and therepeatability is bad. Microarray is a high-throughput detection method,although the detection amount is large and multiple genes can bedetected on one chip at the same time, samples should be labeled byfluorescent dyes, the sensitivity is bad, apparatus are expensive andthe data processing is complex, therefore, it is hard to accuratelycompare the gene expression levels of a given gene from differentsources.

It is a new developed method to determine base sequence bybioluminescence technology^([7-8]). This method is convenient and rapid,and has the advantages of cheap apparatus, low cost and easy to realizeautomation. But this method is limited to analyze the mutation andpolymorphism of genes for it can only detect 10 to 30 basesequences^([9]).

SUMMARY OF THE INVENTION

The purpose of the present invention is to study a method for the assayof relative gene expression level of a given gene from different sourcesby base sequencing technology. That's to say, how to detect the relativegene expression levels by detecting several base sequences, andestablish a convenient method with high sensitivity, accuratequantification and low cost, which can be used in clinical diagnosis.

Technological solutions of the invention are as below, and the detectionprinciple is shown in FIG. 1:

(1) Label a Given Gene from Different Sources by Base Sequencing method.

-   -   This can be realized by two methods. The first method is DNA        adapter labeling method. That's to say, first extract the total        RNA or mRNA of tissues or cells from different sources, and        reverse transcript them into double stranded cDNA, and then use        restriction endonuclease to cut the cDNA from each source into        DNA fragments of different lengths; ligate the cDNA enzymatic        products from each source with DNA adapters that can        differentiate the sources, making the cDNA of each source        labeled with DNA adapters of different sequences. DNA adapters        are composed of two single strand DNA that are not completely        complementary to each other, and its structure is shown in        FIG. 2. That's to say, one of its ends contains sequence 1 that        is complementary to the cut of the above restriction        endonuclease; and it ligate with double stranded cDNA enzymatic        fragments in the action of ligases; the other end is “Y”        structure, which is made up of a pair base sequences 2 and 3        that are not complementary to each other. Sequence 3 is also can        be designed to the one complementary to sequence 2, but in this        case, the 3′ terminus of sequence 3 must be properly modified to        make it won't perform extension reaction in the action of        polymerase. Sequence 2 contains a gene source-specific sequence        4, and sequence 5 that won't change with gene sources is between        this sequence and the 5′ terminus of this strand. Different gene        source-specific DNA adapters can be designed into such state        that the base sequence is only different at sequence 4, but the        type and number of bases forming this sequence are the same.    -   The second method is reverse transcription primer labeling        method. That's to say, first extract the total RNA or mRNA of        tissues or cells from different sources, and reverse transcript        them into cDNA with primers of different sequences, making cDNA        from each source labeled with DNA fragments of different        sequences. The structure of reverse transcription primer is        shown in FIG. 3. Its 3′ terminus (sequence 1 in the Figure) is        composed of multiple thymines, and a gene source-specific        sequence 2 is between the 3′ terminus and the 5′ terminus, and a        base sequence 3 that does not change with gene sources is        between this sequence 2 and the 5′ terminus of this strand.        Different gene source-specific reverse transcription primers can        be designed into such state that the base sequence is only        different at sequence 2, but the type and number of bases        forming this sequence are the same.

(2) PCR Amplify the Same Gene from Different Sources on an EqualProportion Basis.

-   -   Usually, the expression level of the target gene extracted from        tissues is small and it can be detected only by PCR        amplification. One of the key technologies of this patent is how        to amplify the above labeled gene from different sources in a        monotube on an equal proportion basis. First we should design a        gene specific primer (GSP) according to the sequence of the        target gene; meanwhile design another common primer (CP), and        its sequence is the same as the 5 sequence (when labeled with        DNA adapters) in FIG. 2 or the sequence 3 (when labeled with        revere transcription primer) in FIG. 3. In the condition that        primers CP and GSP are present, if some source contains the        target gene then GSP will first anneal with it and extension        reaction occurs, the primer CP anneals with the extension        product and extension occurs; if there is no extension product        of GSP, then primer CP won't extend. A pair of primers CP and        GSP is used to amplify the same gene fragment from different        sources and the T_(m) values of the amplified products are        totally the same (the length and the base species are the same),        so the PCR amplification can be ensured on an equal proportion        basis.    -   If the arm 2 and arm 3 in FIG. 2 are complementary to each        other, then the arm 3 will extend in the action of DNA        polymerase, producing the template for CP annealing. Therefore,        the PCR amplification of the same gene from different sources        cannot be realized on an equal proportion basis.

(3) The Sequencing of the Amplified Products of the Same Gene fromDifferent Sources.

-   -   At present the commonly used sequencing reaction is based on the        principle of gel electrophoresis, which is qualitative        detection, and the quantitative characteristic is bad;        furthermore, it cannot detect the base sequence following the        primer, that's to say, it cannot detect the sequences of the        first 50 bases. Bioluminescent assay is based on PPi detection.        For example, pyrosequencing is a method for sequencing by        orderly adding each dNTP in cycle, which can not only directly        detect the base sequence following the primer, the quantitative        characteristic is also very good, and the number of the        repetitive bases can be determined by measuring the peak height.        The present invention detects the sequences of the amplified        products of the same gene from different sources by sequencing        method based on PPi. The base type in sequence can be used to        differentiate the gene source, and the peak intensity can be        used to judge the gene expression level difference of different        sources. Gene expression level analysis is the important content        of genomics research. The purpose of the present invention is to        apply the sequencing technology to the comparative assay of gene        expression level difference. Compared with the present        technology, the innovation of the invention is: the expression        level difference of the same gene from different individuals can        be detected by only one assay, and additional detection cost is        not required. Easy to be instrumented, laser, gel, fluorescence        labeling, and electrophoresis are not required. This inventive        method has a wide application prospect for its advantages of        high sensitivity, good quantification, low price and simple        operation. This method has a significant meaning for the        screening of disease-related genes, clinical early diagnosis and        the preparation of specific medicine for the treatment of        disease. And it also can be used to study the expression of the        relative genes when human beings, animals or cells are in the        treatment of drugs or other methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the principle chart of detecting the difference of geneexpression level between various sources of the present invention.

FIG. 2 is the structural schematic diagram of DNA adapters.

FIG. 3 is the structural schematic diagram of reverse transcriptionprimer.

FIG. 4 is the sequencing result when using DNA adapters to label the P53genes in human brain cancer tissue, normal tissues and liver cancertissue.

FIG. 5 is the sequencing result when using reverse transcription primersto label the P53 genes in human liver cancer cell and human bladdercancer cell.

DETAIL DESCRIPTION OF THE INVENTION

Concrete examples are used to illustrate the above method, the mainexperimental steps are as below:

(1) Label the same gene from different sources. Respectively extract thetotal RNA or mRNA of relative tissues or cells from each individual anddetect its concentration. Then according to DNA adapter labeling methodor reverse transcription primer labeling method, make the cDNA from eachsource labeled with DNA adapters of different sequences or DNA fragmentsof different sequences. When labeling with DNA adapter labeling method,first reverse transcript the mRNA into double stranded cDNA; cut thecDNA into fragments of a certain length by restriction endonuclease (MobI) that can identify the sequences of the four bases. Respectivelyligate these fragments with the DNA adapters containing genesource-specific sequences. Then mix cDNA fragments of different sourcesthat are labeled with DNA adapters, and use the mixture as the templateof PCR amplification reaction. When labeling with reverse transcriptionprimer labeling method, respectively reverse transcript mRNA into cDNAwith the reverse transcription primers that are corresponding to eachsource, mix after purification, and use the mixture as the template ofPCR amplification reaction.

(2) PCR amplification. Perform PCR amplification reaction on the DNAtemplate in (1) using a common primer (CP) that is not relevant to genesource and a gene specific primer (GSP). Because a pair of primers CPand GSP are used to amplify the same gene from multiple sources, therelative proportion of the given gene in the each source is fixed in theamplification process, that's to say, the amplification is performed onan equal proportion bases, and the proportion won't change with theincrease of amplification times. If the expression level difference ofmultiple genes from the above different sources needs detecting,different GSP needs to be added to perform the amplification.

(3) Quantitatively detect the amplification products of the genefragments from different sources at the same time. After the singlestrand is prepared from the amplified PCR product by biomicrospheretechnique or restriction enzyme digestion, add the common primercomplementary to the template sequence to anneal. Or directly purify thePCR products, and then add the common primer complementary to thetemplate sequence to anneal. Then detect the sequences of two bases bybioluminescence method, namely respectively add dNTP or ddNTPcorresponding to the gene sources to the solution containing substrate.If the added dNTP or ddNTP is complementary to the template, then PPiwill be released. PPi is converted into ATP rapidly in the action ofenzyme, and ATP reacts with luciferin in the action of luciferase toproduce light signal. In the result, the base sequence represents thedifferent gene source, and the signal intensity represents the geneexpression level of each source. According to the gene expression leveldifference of each individual, we can quickly judge the function ofgenes and find the disease-related functional genes.

EMBODIMENT 1

The detection of gene expression level difference of P53 gene in humannormal tissue, brain cancer tissue and liver cancer tissue.

This example detects the expression levels of P53 genes of the tissuesfrom three different sources by DNA adapter labeling method. First,design three different DNA adapters, respectively ligate them with thecDNA fragments digested by restriction endonuclease, and then mix toperform PCR amplification.

1. The Preparation of cDNA Sample.

(1) The extraction of total RNA: respectively get 0.1 g human normaltissue, brain cancer tissue and liver cancer tissue, add 1 ml Trizol tothe Tissuelyser to grind, extract total RNA according to the operationin the Trizol instruction. Identify it by electrophoresis, if the 28 sand the 18 s bands are complete and have no degradation, detect itsconcentration by ultraviolet absorption method, and then regulate itsfinal concentration to 1 μg/μl with sterile DEPC-H₂O.

(2) The synthesis of the first strand cDNA: Oligo(dt)₁₆ (100 μmol/L) 1μl and total RNA (1 μg/μl) 3 μl, the mixture is incubated at 70° C. for10 min, place it on ice, add 4 μl the first strand buffer solution of 5times' concentration, 2 μl DTT (0.1 mol/L), 1 μl Rnase inhibitor (40U/μl), 4 μl dNTP mixture (2.5 mmol/L each), 4 μl DEPC-H₂O, incubate at37° C. for 2 min, add 1 μl Superscript II (200 U/μl), incubate at 42° C.for 1 h, 70° C. for 10 min, cool it down on ice.

(3) The synthesis of the double stranded cDNA: add 30 μl the secondstrand buffer solution of 5 times' concentration, 12 μl dNTP mixture(2.5 mmol/L each), 1 μl E coli ligase (10 U/μl), 10 μl DNA polymerase I(4 U/μl), 1 μl RNase H (2 U/μl) to the above mix solution, add DEPC-H₂Ountil the total volume reaches 150 μl, incubate at 16° C. for 2 h, 70°C. for 10 min.

2. Label the Same Gene from Different Sources.

(1) Enzyme digestion reaction: add 10 μl double stranded cDNA, 2 μlbuffer solution of 10 times' concentration, 1 μl Mbo I TaKaRaendonuclease (10 U/μl), 7 μl distilled water for sterilization, thetotal volume of the reaction system is 20 μl. Place the mixture in 37°C. water bath and react for 2 h, and then place it at 70° C. for 10 minto inactivate the Mbo I enzyme. The feature of the Mbo I endonuclease isthat it can identify the 5′→3′ GATC order in DNA, and cut it to form theGATC adhesive end with the 5′ terminus bumps.

(2) Ligation reaction: get equal volume of endonuclease reactionsolutions of different sources; respectively ligate them with 3different DNA adapters. One strand adp-4 of the three DNA adapters isthe same, another strand contains four gene source-specific bases, andthe four bases only have different sequence, all of them are composed ofc, t, g, and c. Their sequences are: adp-1: 5′-ccc cac ttc ttg ttc tctcat gtca cg cat cac tcg-3′, adp-2: 5′-ccc cac ttc ttg ttc tct cat ctgacg cat cac tcg-3′; adp-3: 5′-ccc cac ttc ttg ttc tct cat atcg cg cat cactcg-3′; adp-4: 5′-gat ccg agt gat gcg cta ag-3′. The parts havingunderlines and italic are gene source-specific bases. adp-1 and adp-4form a DNA adapter 1, adp-2 and adp-4 form a DNA adapter 2, adp-3 andadp-4 form a DNA adapter 3. All of the adapters have the structure withthe 5′ terminus bumps the four bases GATC. DNA adapters 1, 2 and 3 arerespectively used to label the P53 genes in human normal tissue, braincancer tissue and liver cancer tissue. Get 1 μl enzyme digestionsolution, add 2 μl two single strands (10 p mol/L) that form theadapters (2 μl each), 2 μl 10×T4 DNA ligase Buffer and 11 μl distilledwater for sterilization, place the mixture at 70° C. for 10 min, andthen cool down the temperature to 16° C. at the rate of 0.2° C./s, add 2μl T4 DNA ligase (4 U/μl) and react for 2 h.

3. PCR Amplification and the Preparation of the Single Strand.

(1) PCR amplification: mix the above ligation products from threedifferent sources into one reaction tube at the raton of 1:1:1, andrespectively add 2 μl common primer (CP, 5′-ccc cac ttc ttg ttc tctcat-3′) (10 pmol/L), 2 μl specific primer (5′-gga gca cta agc gag cactg-3′) (10 pmol/L) of P53 gene labeled by biotin, 3 μl Mg²⁺ (25 mmol/L),4 μl dNTP Mixture (2.5 mmol/L each), 5 μl 10× PCR Buffer and 0.5 μlTaKaRa Taq DNA polymerase, and then add distilled water forsterilization until the total volume reached 50 μl to perform PCRamplification. The conditions for the PCR reaction are: 94° C. 30 s, 60°C. 30 s, 72° C. 30 s, the reaction is carried out for 35 cycles. Thefinally obtained product is the double stranded DNA labeled by biotin.

(2) The preparation of single strand: get 25 μl M280 beads and wash themaccording to the requirements of operation instruction. Dissolve in 50μl 2× B&W Buffer (wash buffer), add equal volume of PCR product to react30 min, shake slightly in the reaction process so as to make the beadsin a suspension state. Fasten the beads with magnet and desert thesupernatant, add 20 μl NaOH solution (0.1 mol/L) after washing the beads2-3 times with 1× B&W Buffer, and then react for 5 min. Extract thesupernatant to another tube and regulate the pH value to 6˜7 withdiluted hydrochloric acid, and then store it in refrigeration. The solidphase beads are dissolved in the 1× B&W Buffer to store after beingwashed and leave it to use when sequencing.

4. Compare the Relative Expression Level of the Same Gene from DifferentSources by Base Sequence Determination Method.

Prepare the solutions that contain 25 mM Mg²⁺ and 5 mM Tris(pH 7.7) fromthe above single strand DNA sample (the biomicrosphere in step 3), andrespectively add 5 pmol CP into each solution, heat the solutions at 70°C. for 10 min and then naturally cool them down to room temperature. Get1˜5 μl solution and add it into sequencing detection standard mixedsolution of 100 μl, and then orderly add dNTP to perform sequencingreaction.

The composition of the sequencing detection standard mixed solution is:0.1 M Tris-HAc (pH 7.7), 2 mM EDTA, 10 mM Mg (Ac)₂, 0.1% albumin (BSA),1 mM dithiothreitol (DTT), 3 μM adenosine 5′ phosphosulfate (APS), 0.4mg/ml polyvinylpyrrolidone (PVP), 0.4 mM fluorescein, 200 mU/mladenosine triphosphate sulfurylase (ATP-sulfurylase), 2 U/ml apyrase, 1U DNA polymerase Klenow without exonuclease activity.

5. Detection Results.

For DNA adapters 1, 2 and 3 are respectively used to label the P53 genesin human normal tissue, brain cancer tissue and liver cancer tissue,when adding dGTP, the obtained signal intensity represents the geneexpression level from human normal tissue; when adding dCTP, theobtained signal intensity represents the gene expression level fromhuman brain cancer tissue; when adding dATPaS (the analogue of dATP),the obtained signal intensity represents the gene expression level fromhuman liver cancer tissue. The sequencing result is shown in FIG. 4. Thefirst base “C” of the sequence in the Figure is from the DNA adapter 2,representing the expression level A1 of the P53 gene in human braincancer tissue; the second base “G” is from DNA adapter 1, representingthe expression level A2 of the P53 gene in human normal tissue; thethird base “A” is from DNA adapter 3, representing the expression levelA3 of the P53 gene in human liver cancer tissue. The ratio of peakheights of the three base sequences represents the expression leveldifference of the P53 gene in the three sources. The two times'detection results (A1:A2:A3) are: 28.20:24.9:46.9 and 28.1:22.4:49.5,average ratio (A1:A2:A3) is: 28.15:23.65:48.2.

Embodiment 2

The detection of gene expression level difference of P53 gene in humanliver cancer cell and bladder cancer cell.

This embodiment mainly use reverse transcription primer labeling methodto detect the expression level difference of P53 gene in human livercancer cell and bladder cancer cell. That's to say, use primers ofdifferent sequences to respectively reverse transcript mRNA fromdifferent sources, making the cDNA from different sources labeled withDNA fragments of different sequences. And compare the result with theRT-PCR detection result.

1. The Preparation of the Sample to be Detected.

Respectively extract total RNA from human liver cancer cell and bladdercancer cell according to the method in [Embodiment 1]. Identify it withelectrophoresis, if the mass is complete, then detect its concentrationby ultraviolet absorption method, and then regulate its finalconcentration to 1 μg/μl with DEPC-H₂O. Respectively use reversetranscription primers P-1 and P-2 to reverse transcript the mRNA inhuman liver cancer cell and bladder cancer cell into cDNA. The sequencesof reverse transcription primers P-1 and P-2 are: P-1: 5′-ccc cac ttcttg ttc tct cat cag ttt ttt ttt ttt ttt-3′ P-2: 5′-ccc cac ttc ttg ttctct cat gac ttt ttt ttt ttt ttt-3′.

The reaction steps are: get 3 μl primer P-1 or P-2 (10 pmol/L) and 3 μltotal RNA (1 μg/μl), place at 70° C. for 10 min, then place it on ice,add 4 μl the first strand buffer solution of 5 times' concentration, 2μl DTT (0.1 mol/L), 1 μl Rnase inhibitor (40 U/μl), 4 μl dNTP mixture(2.5 mmol/L each), 2 μl DEPC-H2O, incubate at 37° C. for 2 min, and thenadd 1 μl SuperScript™ II RNase H—reverse transcriptase, incubate at 42°C. for 1 h, 70° C. for 10 min, cool it down on ice. Mix at equal volumeafter purification, and use the mixture as the template of PCR reaction.

2. PCR Amplification and the Preparation of Single Strand.

Perform PCR amplification and prepare single strand according to themethod in [Embodiment 1]. Wherein the CP and gene specific primer arethe same as that of [Embodiment 1].

3. Comparatively Assay the Gene Expression Level of the Same Gene fromDifferent Sources by Base Sequence Determination Method.

Prepare the solutions that contain 25 mM Mg²⁺ and 5 mM Tris(pH 7.7) fromthe above single strand DNA sample, and respectively add 5 pamol CP intoeach solution, heat the solutions at 70° C. for 10 min and thennaturally cool them down to room temperature. Get 1˜5 ml solution andadd it into sequencing detection standard mixed solution of 100 ml, andthen orderly add dNTP to perform sequencing reaction.

For reverse transcription primers P-1 and P-2 are respectively used tolabel the P53 genes in human liver cancer cell and bladder cancer cell,so when adding dCTP, the obtained signal intensity represents the geneexpression level from human liver cancer tissue; when adding dGTP, theobtained signal intensity represents the gene expression level fromhuman bladder cancer tissue.

4. Detection Result.

The sequencing result is shown in FIG. 5. The first base “C” of thesequence in the Figure is from the reverse transcription primer P-1,representing the gene expression level A1 of human liver cancer cell;the second base “G” is from the reverse transcription primer P-2,representing the gene expression level A2 of human bladder cancer cell.The ratio of peak heights of the two base sequences represents theexpression level difference of the P53 gene in the two sources. The twotimes' detection results (A1: A2) are: 82.9:17.1, 87.4:12.6, 84.2:15.8,89.5:10.5, average value is: 86:14=6.14:1, standard deviation of thedetection is 3.0:3.0.

The expression levels of P53 gene in liver cancer cell and bladdercancer cell by RT-PCR method are 126359 copies/μl and 22093/μl, theratio is 5.72:1. Compare the detection result of the two methods; therelative average deviation is less then 2%, which indicates that thedetection result of the method in present invention is more accurate.

Indexing in article:

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1. A comparing method for expression amount of the same gene fromdifferent sources by base sequence measurement comprising: (a) use DNAsequence labeling method to make the reverse transcript complementaryDNA (cDNA) of mRNA from different sources contain a segment ofsource-specific sequence; (b) mix the labeled cDNA of different sourcesinto one tube and use it as the substrate of polymerase chain reaction(PCR); PCR amplification is performed using the same common primer and agene-specific primer; (c) Detect the base sequence of the above PCRamplified products corresponding to the gene sources by bioluminescentassay, wherein the base type represents the gene source, and the signalintensity of each base represents the gene expression level from eachsource.
 2. The comparing method of claim 1, wherein the said differentsources mean different tissues or cells.
 3. The comparing method ofclaim 1, wherein the said DNA sequence labeling method means thatrestriction endonuclease is used to prepare double stranded cDNAs fromdifferent sources into fragments of proper length through enzymolysis;and then ligate the fragments with DNA adapters of differentsequences—cDNA from different sources is ligated with different DNAadapters.
 4. The comparing method of claim 3, said DNA adapter containsthe sequence complementing to the cuts of the restriction endonucleasein claim 3, and is composed of two single strand DNAs that are notcompletely complementary to each other; and the adapter can ligate withdouble stranded cDNA enzymatic fragments in the action of ligases. 5.The comparing method of claim 4, wherein one of said two single strandDNAs that are not completely complementary to each other contains asegment of gene source-pecific sequence, and a base sequence that doesnot change with gene sources is between this sequence and the 5′terminus of this strand, and this base sequence is not complementary tothe 3′ terminus of another strand.
 6. The comparing method of claim 1,wherein the said DNA sequence labeling method means that reversetranscript mRNAs from different sources with primers of differentsequences respectively, making cDNAs from different sources labeled withdifferent DNA fragments.
 7. The comparing method of claim 6, wherein thesaid primers of different sequences means that the primer's 3′ terminusis composed of multiple thymines, and there is a gene source-specificsequence between the 3′ terminus and the 5′ terminus, and a basesequence that does not change with gene sources is between this sequenceand the 5′ terminus of this strand.
 8. The comparing method of claim 1,wherein the said common primer means that the primer's sequence ispartly or completely similar with the base sequence (as mentioned inclaim 5 and claim 7) that does not change with gene sources.
 9. Thecomparing method of claim 1, wherein said bioluminescent assay means themethod that quantitatively assay the pyrophosphate (ppi) produced byextension reaction.
 10. The comparing method of claim 9, wherein saidextension reaction means: use the PCR amplified product of claim [1] orits single strand product as template, add the sequencing primers toanneal, then orderly add dNTP, or ddNTP or their analogues, in theaction of DNA polymerase, when the added dNTP or ddNTP or theiranalogues complementary to the template, the polymerization occurs.